Glutathione reductase modulates endogenous oxidative stress and affects growth and virulence in Avibacterium paragallinarum

Abstract

Glutathione reductase (GR) plays a pivotal role in managing oxidative stress, a process crucial for microbial virulence and adaptation, yet it has not been extensively explored in bacteria such as Avibacterium paragallinarum (Av. paragallinarum). This study examined the specific roles of GR in Av. paragallinarum, focusing on how GR modulates the bacterium's response to oxidative stress and impacts its pathogenic behavior. Using gene knockouts together with transcriptomic and metabolomic profiling, we identified an important shift in redox balance due to GR deficiency, which disrupted energy metabolism and weakened the oxidative stress defense, culminating in a notable decline in virulence. In addition, decreased growth rates, reduced biofilm production, and weakened macrophage interactions were observed in GR-deficient strains. Notably, our findings reveal a sophisticated adaptation mechanism wherein the bacterium recalibrated its metabolic pathways in response to GR deficiency without fully restoring virulence. Our in vivo studies further highlight the pivotal role of GR in pathogen fitness. Together, our findings connect GR-mediated redox control to bacterial virulence, thereby furthering the understanding of microbial adaptation and positioning GR as a potential antimicrobial target. Our insights into the GR-centric regulatory network pave the way for leveraging bacterial redox mechanisms in the development of novel antimicrobial therapies, highlighting the importance of oxidative stress management in bacterial pathogenicity.

Keywords: Av. paragallinarum; Bacterial virulence; Glutathione reductase; Metabolic adaptation; Oxidative stress; Redox homeostasis.

Figure 1

GR knockout in Av. paragallinarum affects growth and host–pathogen interactions. A Schematic diagram showing the construction strategy for the GR knockout mutant in Av. paragallinarum using a kanamycin resistance cassette (Kan). The schematic outlines the homologous recombination process used to replace the GR gene with the Kan cassette, detailing the steps for successful gene transfer and integration into the bacterial chromosome. B PCR analysis and product sequencing confirmed the knockout of the GR gene. Lane 1 represents the wild-type strain (WT, 1798 bp), lane 2 represents the positive control with the plasmid containing the kanamycin resistance gene (1258 bp), and lane 3 represents the GR knockout strain (ΔGR, 1258 bp). C Western blot analysis was used to show the absence of GR protein in the GR mutant (ΔGR) strain compared to the wild type (WT) strain. The target protein size is approximately 48.75 kDa. The absence of a band in the ΔGR lane indicates successful GR gene knockout, while the presence of a band in the WT lane confirms the expression of the GR protein. D Growth curve comparison of WT and ΔGR strains under aerobic conditions. E Quantitative assessment of biofilm formation in WT and ΔGR strains. F The macrophage interaction assay was used to assess the adhesion capability of WT and ΔGR strains over time. G The invasion assay was used to evaluate the invasion capabilities of WT and ΔGR strains into HD11 chicken macrophages.

Figure 2

In vivo pathogenicity studies reveal diminished virulence in GR-knockout Av. paragallinarum . ASchematic showing the design of the animal virulence assay. Chickens were subcutaneously inoculated with different doses of Av. paragallinarum strains in the infraorbital sinus and monitored for 7 days. B Disease severity in chickens infected with different doses of the ΔGR strain compared to the WT strain. C Disease incidence in chickens exposed to different doses of the GR mutant (ΔGR) strain compared to the wild-type (WT) strain (P < 0.0001). D The disease severity categorization model was used to determine virulence levels.

Figure 3

Transcriptomic characteristics and functional annotation before and after GR knockout in Av. paragallinarum. A PCA was used to compare the transcriptional profiles of wild-type (WT) and GR-knockout (ΔGR) strains. B Identification of 211 DEGs including123 upregulated and 88 downregulated genes. C Heatmap showing the cluster analysis of DEGs and the distinct expression profiles of upregulated and downregulated genes in ΔGR strains. D GO enrichment analysis of the DEGs shown as a bar graph. E Detailed GO enrichment analysis of the upregulated and downregulated DEGs. F Comparative analysis of metabolic pathways.

Figure 4

Confirmation of oxidative stress-related differential gene expression changes in GR-knockoutAv. paragallinarumby RT-qPCR. A RT-qPCR was used to compare GR mRNA expression levels in wild-type (WT) and GR-knockout (ΔGR) strains (P < 0.0001). B–I RT-qPCR analysis of several metabolic genes including gnd (P < 0.0001) (B), PxpA (P = 0.0035) (C), HBL79_RS05810 (P = 0.0005) (D), HBL79_RS03605 (P = 0.0041) (E), EIIB (P = 0.0010) (F), GPx (P = 0.0007) (G), HBL79_RS11455 (P = 0.0302) (H) and HBL79_RS03835 (P = 0.0013) (I).

Figure 5

LC–MS/MS metabolomics analysis unveils alterations in metabolic functions and pathways in Av. paragallinarumassociated with GR knockout. A PCA was used to compare the metabolic profiles of wild-type (WT) and GR knockout (ΔGR) strains of Av. paragallinarum. B Annotation of top differentially regulated metabolites. C Variable importance in projection (VIP) and fold change (FC) analysis of differentially regulated metabolites between WT and ΔGR strains. D Correlation heatmap displaying the coordinated metabolic response to GR knockout. E KEGG enrichment analysis of differentially regulated metabolites between WT and ΔGR strains of Av. paragallinarum.

Figure 6

Analysis of oxidative stress metabolites inAv. paragallinarumfollowing GR knockout. A GSH/GSSG ratio in wild-type (WT) and GR-deficient (ΔGR) strains of Av. paragallinarum (P = 0.0011). B NADP + /NADPH ratio in WT and ΔGR strains (P = 0.0021). C. NADH/NAD + ratio in WT and ΔGR strains (P = 0.0002). D Total GPx ratio in WT and ΔGR strains (P = 0.0099). E Selenium-dependent GPx ratio in WT and ΔGR strains (P = 0.0096). F Non-selenium-dependent GPx ratio in WT and ΔGR strains (P = 0.0677). G Total ROS ratio in WT and ΔGR strains (P = 0.0016). H Intracellular ROS ratio in WT and ΔGR strains (P = 0.0005). I Supernatant ROS ratio in WT and ΔGR strains (P = 0.0247).